Molecular tag reader

ABSTRACT

Near infrared molecular assays can be used to detect small quantities of a molecule of interest in vivo or in vitro using laser dyes. A hand-held portable device is provided which can rapidly read small quantities of selected molecular tags in tissues of fish or other animals in the field. The device is composed of four components:
         (1) a light source, such as a laser diode;   (2) a sample holder;   (3) an optical system; and   (4) a detector.

FIELD OF THE INVENTION

The present invention relates to a hand-held electro-optical systemwhich can rapidly read small quantities of selected molecular tags intissues of animals.

BACKGROUND OF THE INVENTION

There has long been a desire to develop a universal system for detectingmolecular species found in biological fluids or synthetic chemicalenvironments. Currently there are a variety of specialized instruments,all of which are large, cumbersome, relatively slow, and expensive tooperate, requiring a highly skilled staff.

Two of the most commonly used techniques for measuring the presence andquantity of an analyte in a test sample are ELISA and RIA.

There are many different types of ELISA procedures. The most generalformat consists of depositing the antigen of choice at a specificconcentration in a 96 well plastic plate. The antigen solution isincubated in the plate for one hour before excess antigen is washed out.The plate is then coated with antigen, but the remaining electrostaticcharges on the plate must be blocked with a protein buffer for anotherhour so that test proteins and reagents, which are added later, do notnon-specifically bind to the plate. The plate is washed once againbefore adding the test samples and incubated for another hour. Thiscycle of washing is repeated and an enzyme labeled anti-ligand is addedand incubated for one more hour. Once this last incubation is finished,the plate is thoroughly washed and the enzyme substrate is added. Ifenzyme is present, the substrate will be converted to product. Acolorimetric change occurs which is measured by suitableinstrumentation. The data are then expressed on a computer screen orprinted by a printer. In some ELISAs, the sample preparation may take asmuch as an entire day before a reading can be taken.

An RIA is usually more sensitive than an ELISA. The probe used isradioactive and requires special disposal facilities. The sequence stepsof the assay are the same as the ELISA, but the probe binds directly tothe target molecule without enzymatic conversion of a substrate to acolored product. In a competitive RIA, the radioactive probe and thenon-radioactive molecule of interest found in the test sample competefor a common binding site. The gamma and beta radioactive counters usedto detect the radiation are large table top or floor model instrumentsthat print out data.

Other techniques and complementary instruments are used for detectingbiomolecules, including PANDEX, TDX, HPLC (high performance liquidchromatography), PHAST, GC (gas chromatography), FACS, and others.

Unfortunately, none of the above assays is capable of providing a rapid,reliable identification of fish or other animals rapidly and accuratelyas to their age, origin, and any experimental protocols to which theyhave been subjected. Presently available methods are cumbersome (e.g.,wire tags), lethal (e.g., otolith marks and wire tags), expensive, andtechnically demanding (e.g., rare earth metals and genetic testing). Allof these methods are time consuming, and most are too large to use withvery small fish.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome the aforesaiddeficiencies in the prior art.

It is another object of the present invention to provide a tool toidentify stocks of fish or other wildlife without endangering them.

It is another object of the present invention to provide a method andapparatus for quick and easy identification of marked fish or otheranimals.

It is a further object of the present invention to provide a device thatcan be used in the field environment to identify fish and other animalsrapidly and accurately as to their age, origin, and experimentalprotocols to which they have been subjected.

It is another object of the present invention to provide a means foridentifying commercial livestock herds.

It is yet another object of the present invention to provide a means foridentifying valued animals such as horses and dogs.

According to the present invention, a hand-held portable device isprovided which can rapidly read small quantities of selected moleculartags in tissues of fish or other animals in the field. The device iscomposed of four components:

-   -   (1) a light source, such as a laser diode;    -   (2) a sample holder;    -   (3) an optical system, made of a fiber optic lens and a bandpass        filter; and    -   (4) a photo diode detector coupled to an LCD.

The optical system can be modified to eliminate the fiber optics andbandpass filter.

The laser may be replaced by a miniature light bulb coupled to an extrabandpass filter to eliminate lower light wavelength that might interferewith the sensitivity of the instrument. The light sources and othersystems can be battery operated.

The optical system is optimized for the type of tag used in the markingprocedure. Settings for the optical system are selected based upon thedyes used for tagging. Multiple dyes in one organism can be tested bychanging the excitation wavelength and emission measurement settings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a device according to the present invention for detectingmolecular markers.

FIG. 2A shows one embodiment of a sample analyzer head.

FIG. 2B shows the fiber optic arrangement.

FIG. 3 shows a second embodiment of a sample analyzer head.

FIG. 4A shows a side view of an instrument designed for detectingmolecular markers in eels.

FIG. 4B shows a top view of an instrument designed for detectingmolecular markers in eels.

FIG. 4C shows a cutout view of an instrument designed for detectingmolecular markers in eels.

FIG. 5A shows a side view of disposable tip.

FIG. 5B shows a side view of another disposable tip.

FIG. 5C shows a pressure fitting for the disposable tip of FIG. 5B.

FIGS. 6A, 6B, and 6C show a device in which detection is accomplishedwithout an immobilizing matrix or reaction vessel wall.

FIG. 7 is a schematic of the process for detecting molecular markersaccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

As shown in FIGS. 1, 2A, 2B and 3, a small (0.5 W) laser diode 101 withan emission wavelength of 785 nm±5 nm operating on its own 6 volt powersupply is mechanically coupled to the sampler analyzer head 102. Thesample analyzer head 102 is a block of machined hard aluminum or TeflonPTFE, which holds the optic housing 103, the bore 104 for the disposabletip or tube 105, and a negative/positive pressure connecter 106. Theoptic housing 103 includes a narrow band pass filter 107 of 850±25 nmand a plano convex lens 108, which may be made of BK7 glass. The optichousing 103 is linked through a fiber optic 109 to a silicon photodiode-amplifier 110 with a spectral peak of 740±50 nm, which is wired toan LCD voltage display 111 of 0.05% accuracy in 1 volt increments.

The pressure connector 106 of the sample analyzer head 102 is attachedto a stepper motor syringe (piston) assembly 112 with a short tube 113.The electrical power for the stepper motor 114 is activated by a motorcontrol board 115 with a variable speed control switch. The total systemis synchronized by a master power and an electronic timer control board116. The photo diode 110 can be replaced by a photon counting module formore sensitive emission measurements.

As shown in FIG. 3, the level of fluid in the reservoir and uptakechannel is determined by timers or by two miniature sensors 117 (sonic)in the sampler analyzer head which are connected to the stepper motorboard 115.

FIGS. 4A–4C show another embodiment of the present invention which canbe used for monitoring eels. In this embodiment, an eel is introducedinto the eel sample tube channel 123 and subjected to light from a laserdiode 120, the wavelength of the laser depending upon the fluorescentdye used. The sample analyzer head and the detector are in one block,121, which comprises the optical power detector wand 122 that attachesto a hand-held optical power meter (not shown). The wand should read ina narrow or specific wavelength corresponding to the wavelength emitted,or with a narrow band bass filter set, or mechanism that would selectspecific wavelengths before its detector window, since the wand can becalibrated to measure only light emitted at a predetermined wavelength,depending on the fluorescent dye used. This can be done with a narrowpass filter grading system or the like.

The detector measures the light emitted from the laser dye in the fishin microwatts. An increase in the microwatt readings corresponds to anincrease in the amount of light emitted, and hence corresponds to a setamount of laser dye in the sample or fish tested.

The eel sample tube channel 123 can have varying diameters toaccommodate various size of fish. The eel sample tube channel 123 ismanufactured with a large diameter that can be reduced by inserting aseries of tubes with smaller internal diameters to accommodate thecorrect sample size.

Eels passing through the sample tube channel 123 can be counted bybreaking a light path 124 as they flow through the channel. The electriceye is placed at the entrance of the sample analyzer head 121. Eelsflowing through the tubing can activate the instrument.

Detection Methods

To detect animals, the laser dye is coupled to a labeling molecule suchas an antibody or a specific ligand such as avidin, Protein A, ProteinG, various antigens, or solid materials such as ion exchange resins,latex beads, or other inert non-fluorescent structures, and use thislabeled compound as the reagent tag, i.e.,DYE+LABELING MOLECULE OR SOLID MATERIAL=REAGENT TAG

The standard detection techniques are then carried out as described inFIGS. 1–5. FIG. 6 is a special application in which detection isaccomplished without the interference of any immobilizing matrix orreaction vessel wall. This special detection system eliminates anybackground emission from materials other than the tag, and hence it ismore sensitive than the matrix dependent method. By lowering thebackground to near zero, the measurements are more accurate and precisesince they do not depend on the quality of materials used in theproduction of disposable tips. The results are expressed in positivevalues rather than the negative correlations seen in competitiveradioimmunoassays (RIA's). In a competitive RIA system, the amount ofradioactive label seen or counted decreases as the target molecule beingdetected increases. In the Near Infrared Molecular Assay (NIRMA) of thepresent invention, an increase in level or concentration of the targetmolecule directly corresponds to an increase of dye, and hence anincrease in light emission.

The Reagents

Among all spectrofluorescent methods, laser fluorimetry is the mostsensitive (1–6). Sauda et al., (1986) demonstrated detection of 10⁻¹² Mlaser dye with a 3 nW laser. Also, there are over 2000 laser dyescommercially available (7) from which one can choose to develop apractical detection system. Therefore, a diode laser, a photo diode, anda laser dye must match in order to make the detection system workable.

Since most biological and other materials do not fluoresce between 700and 1300 nm (4), it was important to find a useful laser dye in thatrange which could bind to a specific carrier or labeling molecules andact as a tag without losing its fluorescent properties.

Dyes for use in the present invention must have at least the followingcharacteristics:

-   -   (1) must fluoresce between 700 and 1300 nm    -   (2) must be soluble in water    -   (3) must be non toxic    -   (4) must be stable when coupled to proteins including during        cold storage    -   (5) must bind electrostatically to specific proteins such as        albumin, lipoproteins, and gamma globulins.

While polymethine dyes have an absorption range between 600 and 900 nm(4), most are positively charged and thus are not very useful fordirectly labeling proteins unless the proteins are negatively charged.Some laser dyes, such as IR-125 and IR-144, are negatively charged, andhence ideal for labeling positively charged proteins (8). A new dye,NN382, can also be used since it has good labeling characteristics.

NN382 has been found to be particularly useful for use in molecularmarking of animals. This dye has the following formula:

The molecular formula of NN382 is C₄₅H₄₈N₃O₁₃S₅Na₃, the compound has amolecular weight of 1067.

The laser dye NN382 has the following characteristics:

-   -   (1) absorption maximum of 778 nm    -   (2) emission maximum of 806 nm    -   (3) soluble in DMSO and related organic solvents    -   (4) soluble in pure water    -   (5) polymerizes and quenches at high temperatures    -   (6) non toxic    -   (7) can be stored frozen for long term storage    -   (8) stable when coupled to proteins and stored in the cold        (below 5° C.) and    -   (9) binds electrostatically to specific proteins such as        albumin, lipoproteins, and gamma globulins.

Because wildlife that is to be detected using the system of the presentinvention is by definition outdoors, the system, including dyes usedtherein, must be operable under a variety of conditions and at leastfrom about 5° C. to about 20° C. For purposes of the present invention,“cold” refers to temperatures below about 5° C. Of course, thetemperatures at which assay are conducted depends on the type of sampleused. Water-based samples are generally assayed at temperatures aboveabout 0° C. so that they are in the liquid state.

Disposable Reaction Tips

The present invention can use two fundamental disposable tipconfigurations, which are defined by two different types of analysistarget area (ATA). The first type, type 1, has an enclosed ATA composedof a solid phase. This type, 130, is shown in FIGS. 5A–5B. The secondtype, type 2, 150, has an ATA which is solid phase free and not enclosedby materials, as seen in FIGS. 6A–6C.

In tip type 1, 130, the material used for the wall of the disposable tip131 has a low near infrared excitation/emission profile in thewavelength of interest. One example of a useful material is Teflon FEP,although other materials can be used. Of suitable materials, Teflon FEPis preferred. Many other materials have a higher near infraredbackground or other less appealing characteristics, such as carryinglarger electrostatic charges on the surface and hence attracting andbinding biomolecules of interest or reagent tags, which renders theassay less sensitive. The electrostatic charges of the tip must bespecially blocked without interfering with the binding characteristicsof the plug or matrix, 132. Also, since the tips can be used in asterile environment, they must be sterilizable. This is preferablyeffected by radiation, and must not change the properties of any of thematerials used in constructing these tips so that the assay is notaffected in any way.

The analysis target area (ATA) 133 can be made of micro ground glass,micro glass, or plastic beads, as well as porous nylon matrix, porouscomposite matrix (e.g., nylon and ion exchange resins) or a fine mesh orscreen surface. These materials have the same near infrared, lownon-specific binding and sterilizable properties as the FEP housingabove. Also, they have an added characteristic, in that they can bindcovalently or by other means “target” or “capture” molecules used in thesystem of the present invention. The target molecules are the chemicalstructures which are bound to the matrix for the purpose of havingspecific molecules recognize and bind to them when they are in veryclose proximity. For example, the target molecule could be an antigen,an antibody, a ligand such as avidin, concanavalin A, protein A, proteinG, etc., or a hapten, such as biotin, an enzyme substrate or itsproduct, a chelate, etc. The matrix may not carry any target or capturesmolecules at first, but these may be supplied in an activated form sothat the user can attach any ligand of choice to the matrix.

FIG. 5A shows an FEP tube 140 (24 gauge) with a plug 132 composed ofactivated ground glass sandwiched between two plastic porous (porex)discs 141, 142. The tube 140 fits into a pressure fitting with an “O”ring 143 to seal the tube.

In FIG. 5B, the reservoir 144 is larger than the uptake channel 145,permitting a bigger sample volume to be collected and tested. In thisembodiment, internal pressure fittings 146 are used rather than anexternal one with an “O” ring 147 as shown in FIG. 5C.

FIG. 6A shows a type 2 disposable tip 150 which has the same externaldisposition as the tip in FIG. 5B except for a double window in frontand back of the ATA 156. Internally, the tip 150 has a small bore tube151 specially designed to bring a small microliter bubble 152 in frontof the detection window. The tube end 153 is designed so as not to touchthe internal wall of the tip. Also, by beveling the tube 151, oneincreases the surface area for better surface adherence of the bubble.

Another embodiment of this design is shown in FIG. 6B in order toflatten out the bubble 155 in which detection occurs. In thisembodiment, a post 158 is set close to the tube end 157. In this case,the bubble 155 is hung between the tube end 157 and the post 158, henceincreasing the stability of the sample and making surface readingspossible with less scatter of the emission wavelength from the reagenttag. A space 159 exists on each side of the post between the post andthe internal wall of the tip 160 so that air has access to the uptakechannel. When a negative pressure is created, the fluid sample can risethrough the uptake channel into the reagent trap and past it to the ATA.

These type 2 disposable tips have a “trap”, which is a zone where twocompeting molecules bind to a matrix. The trap is situated before theend of the specially beveled tube as shown in FIGS. 6A and 6B.

Methods of Operation

FIG. 7 illustrates an antibody determination according to the presentinvention. A disposable type 1 tip 201 has a specific antigen Z1 boundto its matrix. This is done by using covalent coupling chemistries,although any other means of binding the antibody to the matrix can beused. A fluid sample containing the specific antibody “Z” to antigen“Z1” is aspirated though the uptake channel past the matrix The antibodyZ binds to the antigen Z1. Next, the sample fluid is stopped at apredetermined level inside the disposable tip so as not to contaminatethe sample analyzer head. The sample is then expelled from the tip. Aslong as the antigen Z1 is not saturated by an initially highconcentration of antibody Z, the antibody Z binds to the antigen Z1during this phase as long as there is more antibody Z available forbinding. A wash buffer is then flowed through the matrix to wash off thesample, as in 3 and 4 above. Next, a specific reagent tag is taken up toa determined level past the matrix. The reagent tag in this case is anantibody tag binding to any antibody Z present. The amount of antibodytag binding to antibody Z determined the intensity of the emission. Ifno antibody Z is present in the sample, no antibody Z will bind toantigen Z1, and hence no antibody tag will be in the analysis targetarea once there is no antibody Z to bind to. The reagent tag isexpelled. The unbound reagent tag is flushed out of the matrix as above,but the matrix is maintained wet to enhance the reading. The diode laseris fired for a predetermined amount of times and the photo diode picksup the quantity of light emitted by the reagent tag. The voltagegenerated is amplified and displayed on the liquid crystal display. Thenumber displayed represents a known quantity of antibody Z.

In another embodiment of the present invention, the reagent tag is anavidin tag binding to a biotinylated antibody, which is attached to aspecific antibody to the antigen (Ag2) coupled to a solid matrix.

In this embodiment, an antigen determination is conducted. Themechanical sequence of events is identical to those for detecting anantibody. The molecular interactions between antibodies are bound to thematrix rather than to the antigen. This can be effected by directbinding of the antibody to the matrix or through linkers and spacers.One type of spacer molecule is avidin. Coupling of avidin to the matrix,followed by binding of a specific biotinylated antibody Xa as thecapturing molecule represent a universal coupling technique useful inantigen detection. This antibody Xa recognizes and captures antigen X2,which is then recognized and bound by a second antibody reagent tag Xb.Diagram 3 illustrates multiple variations of this theme.

In another embodiment, the antigen Ag1 is captured by the matrix boundAG1 specific antibody. The antigen is then recognized and bound by asecond biotinylated antibody, which is then bound by the avidin reagenttag.

Antibody or antigen determinations can also be made by reagent tagscomposed of near infrared probes in or on latex particles, ion exchangeresins, or other particulate matter with the appropriate biophysical andchemical characteristics, as shown in Diagram 5.

FIG. 2B illustrates detection of emitted light from the reagent tag. Thelaser diode 101 excites the dye NN 382 at 785 nm, which is very close tothe maximum excitation absorbance of the laser dye, i.e., 778 nm. Thelaser light hits the matrix 132 in the disposable tip 150 at a 45 degreeangle, but the detector lens 108 and the filter 107 are at a right angleto the emitting matrix. It is important to note that the matrix 132 isnot transparent, and therefore the light must be detected on the sameside as the excitation in order to capture most of the signal from thematrix. Also, the excitation angle prevents most of the reflected lightfrom entering the detection system, since it bounces off at a 45 degreeangle away from the lens. The filter stops the laser light from enteringthe optical housing 170 and only lets emission light pass through. Theemitted light from the dye on the matrix 132 is focused on the end of afiber optic 160 contained in a fiber optic housing 161 by a plano-convexlens 108 closely situated to the emission source. The closer the lens isto the source, the more light it can capture. A large lens with a shortfocal length would be ideal. However, if the optical housing 170 is tooclose to the emission source, it infringes on the laser light path. Theoptical housing 170 and the sample analyzer head chambers are coatedwith a black matte non-reflecting or emitting paint.

Assays described above are based upon type 1 disposable tips in whichthe reagent tag is on a matrix surrounded by the tip wall. Another typeof assay is based on type 2 disposable tips, which increases thesensitivity and decreases error in the assay.

With a type 2 tip, a ligand is coupled to the matrix, which ligandspecifically binds the molecule of interest in a test sample as well asthe reagent tag. The reagent tag is the same as the molecule of interestbut is labeled with a laser dye, such as NN 382. This is a competitiveassay in which an antibody b coupled to the matrix specificallyrecognizes and binds the test sample antigen b1 flowing through thematrix, as well as the reagent tag, antigen b1-NN 382. In order tomeasure the amount of antigen b1 in a test sample, a calibrated quantityof reagent tag is mixed with the test sample. The amount of reagent tagadded to the test sample is just enough to saturate all of the matrixbound antibody b binding sites when no other competitive antigen to thereagent tag is present in the sample being tested. Hence, no excessreagent tag flows through the matrix, as it is all captured by theantibody on the matrix and appears in the ATA, giving a zero reading.The only means by which the reagent tag appears in the ATA is when thereagent tag and antigen b1 from the test sample are mixed. Test sampleantigen b1 competes against the reagent tag for the same binding site ofantibody b located on the matrix, and therefore some of the reagent tagis not bound to the matrix. The free reagent tag flows into the ATA,where it is measured.

As increasing amounts of test sample antigen are present in the mixture,more and more of the reagent tag appears in the ATA. This method showsthat, with a very low concentration of test sample antigen b1, oneobtains low readings, while, with equal amounts of reagent and testantigen b1, half the reagent will appear in the ATA bubble. Finally,when high concentrations of antigen b1 are tested, most of the reagenttag will be found in the ATA. Graphic representation of the NIRMA vs.RIA is shown as the last part of diagram 6. In the NIRMA graph, it canbe seen that as the concentration of antigen in the sample rises, thereagent tag readings increase proportionately. In the standardcompetitive RIA, the converse is true: as the concentration of theantigen in the sample increases, the amount of radioactive tag beingmeasured decreases. RIA requires generation of a standard curve, whileNIRMA does not, since it is established that a certain amount of antigenbinding the matrix will displace an equal amount of reagent into theATA.

Details of measurements taken in the ATA of the disposable tip type 2can be seen in FIG. 6C. The exciting laser light 200 is focused in thecenter of the bubble 171, which is the ATA, while the emissionwavelength from the bubble is captured by the detector lens 108 withoutany interference from building materials used in making the tip.

When using the eel laser instrument, shown in FIG. 4, the eel (notshown) is passed through the eel sample tube 123. The eel is alignedwith the ATA, the laser is fired, and the amount of dye in the eel ismeasured from its emission by the optical detector wand 121. The resultsare expressed in mW.

Near infrared molecular assay, or NIRMA, provides speed of operation, inwhich animals can be detected in seconds versus hours of days. The assaycan be used with small samples, from less than about one microliter, toabout 1000 microliters. The assay is non-isotopic and non-enzymatic, anduses a small, portable, hand held tool which can be battery operated.There is no fluorescent background because of the selection of the nearinfrared range of the reagent tag. The assay is highly sensitive,because of the use of near infrared, two types of tips, and theelectro-optic designs. There is no requirement for precalibration or forproviding a standard curve. The system is intuitive, and little trainingis required. The assay can be used under sterile and non-sterileconditions. By selecting the tip and optical system used, the assay canbe adapted to low concentrations of molecules in a test sample. Comparedto other methods for detecting animals, the method of the presentinvention is not expensive. The tags cannot be removed by poachers, andthe assay system can be used to encode complex information over longperiods of time, i.e, years.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without departing from the generic concept,and, therefore, such adaptions and modifications should and are intendedto be comprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology employed herein is for the purpose of description and not oflimitation.

REFERENCES

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1. An apparatus for detecting a target molecule in a sample fluid invivo or in vitro, comprising: an uptake channel enclosed by a wall andhaving a receiving end to receive the sample fluid; a matrix within theuptake channel, the matrix having a capture molecule for the targetmolecule immobilized therein; an inner tube connected to the matrixopposite the receiving end of the uptake channel; a reservoir on a sideof the matrix opposite the receiving end of the uptake channel, andformed by a wall extending from said wall of said uptake channel, saidreservoir wall further extending beyond and surrounding said inner tube;an analysis target area in said reservoir extending from an end of theinner tube opposite the matrix, wherein a bubble from the sample fluidcan be formed therein; a reagent tag that binds to the target moleculeand fluoresces when subjected to near-infrared light emissions; a lightsource focused directly on the analysis target area where the bubble isformed, the light source configured to emit light in a wavelengthcomprising near-infrared light emissions; and a detector configured todetect the reagent tag that fluoresces within the analysis target areawhen subjected to the light source.
 2. The apparatus according to claim1 wherein the light source is a laser diode.
 3. The apparatus of claim 1wherein the detector comprises a fiber optic lens and a bandpass filter.4. The apparatus according to claim 1 wherein the detector comprises aphotodiode coupled to an LCD.
 5. The apparatus of claim 1 wherein thematrix comprises one of micro-ground glass, micro-glass, plastic beads,nylon, a mesh, and a screen, and the matrix has physical barriers onopposite sides thereof.
 6. The apparatus according to claim 1, whereinthe reagent tag comprises a laser dye.
 7. The apparatus according toclaim 6, wherein the laser dye is soluble in water and bindselectrostatically to one or more of albumin, lipoproteins, and gammaglobulins.
 8. The apparatus according to claim 7, wherein the laser dyecomprises a negative charge.
 9. The apparatus according to claim 8,wherein the laser dye has the formula C₄₅H₄₈N₃O₁₃S₅Na₃.